As4.1 cells (ATCC #CRL2193), a renin-expressing cell line of renal origin that we developed by transgene-targeted tumorigenesis, are being used in concert with transgenic analyses in vivo to elucidate mechanisms governing transcriptional regulation of mouse renin genes. This proposal refines and tests a mechanistic model derived from current data which suggests that renin expression is achieved through cooperation of cell- specific and general factors binding at a crucial, proximal promoter element and distal enhancer element(s) to abrogate or override negative regulatory elements that are ubiquitously functional. Transgenic reporter assays, utilizing EGFP, indicate that all regulatory information required to correctly specify known patterns of renin spatially, temporally, and in response to physiological perturbation, are resident within 4.1 kb of the 5' flanking sequence of the mouse Ren-1c gene. Under the Specific Aims, a variety of approaches will be used to examine in detail our finding that Ren genes are immediate downstream targets of Class 1 Hox genes acting through a proximal promoter """"""""switch-like"""""""" element. Transgenic and cellular transfection assays will be used to assess whether a single complex, or multiple discrete, enhancer(s) are differentially driving expression at key sites of expression observed in vivo such as the renal vasculature and placental tissue. Cis-acting elements observed to mediate potent downregulation of renin expression in As4.1 cells in response to cytokine, hormone and mechanical distention will be mapped within the 5' flanking sequence by in vitro transient-transfection assays prior to testing involvement in vivo by transgenic analysis. Current data suggest that cis-acting elements identified as critical to mediating high level expression of renin in As4.1 cells are, in fact, utilized in vivo. Moreover, the major elements appear to be conserved evolutionarily across species, including human. Identification of the specific recognition sequences, their cognate protein partners and elucidation of the mechanism of interaction, should provide fundamental knowledge on the role of the renin-angiotensin system in normal physiology and abnormal pathophysiological states, as well as afford insight into its role in vertebrate evolution.
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